Psoriasis is one of the three most prevalent autoimmune diseases and has a global morbidity of about 3%, with one third of patients showing moderate to severe conditions. Psoriasis symptoms manifest in skin thickening, extensive lesions, could cause itching, scaling and pain, and have significant influence on the quality of life, mental health and social relationships of patients. In addition, patients having more severe conditions are more susceptible to complications such as arthritis, heart diseases and diabetes, and even risk of death.
1 Autoimmune Psoriasis
1.1 Advances in Pathogenesis Research on Autoimmune Psoriasis
Until 1980, the researchers had paid only attention to the apparent psoriatic symptoms of psoriasis and believed that psoriasis was caused by excessive proliferation of skin keratinocytes. In the subsequent 20 years, with the development in immulogy, researchers detected in the skin of psoriasis patients significant increase of p40 protein, which was considered only a subunit of IL-12p35p40 at the time with IL-12p35p40 being an important factor driving the differentiation of naive CD4+ T cells into Th1 cells. Therefore, scholars at that time recognized psoriasis as an inflammatory disease mediated by abnormal activation of Th1 cells. However, in 2000, Oppmann B et al. discovered the p19 protein, which might polymerize with p40 to form IL-23p19p40. Thus, researchers realized that the theory of Th1-mediated psoriasis proposed on the basis of the difference in p40 expression was flawed. Consequently, Lee E et al. found upon further investigation that IL-23p19 and the p40 protein were both significantly increased in the skin of psoriasis patients but IL-12p35 was not significantly changed. In around 2005, Langrish C L, Harrington L E and Park H et al. found that IL-23 could promote the development of naive CD4+ T cells towards Th17 which regulated the inflammatory response mainly through the secretion of IL-17A. Currently, Leslie van der Fits et al. have demonstrated that autoimmune psoriasis was mediated primarily by the IL-23/IL-17A inflammatory axis (van der Fits, L. et al., Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. Journal of immunology (Baltimore, Md.: 1950) 182, 5836-5845, doi:10.4049/jimmunol.0802999 (2009)). The pathogenetic process of this disease generally involves the following: pathogenic factors (e.g., genetics, environment, infection and physical lesions, etc.) induce the secretion of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 from innate immune cells (e.g., keratinocytes, natural killer cells), which then activates innate immune cells such as dendritic cells; the activated dendritic cells migrate to immune organs such as skin lymph nodes, present antigens and secret pro-inflammatory cytokines such as IL-23, promoting the differentiation of naive CD4+ T cells into Th17; the differentiated self-reactive Th17 migrates out of the skin capillaries, infiltrates the skin at the inflammatory sites; upon re-stimulation by autoantigens, it resumes proliferation and secrets many types of inflammatory cytokines such as IL-17A; the cytokines such as IL-17A can activate keratinocytes and stimulate their proliferation, leading to psoriasis symptoms; on the other hand, the activated keratinocytes can also secrete antimicrobial peptides (such as LL-37 antimicrobial peptide and β-defensin), pro-inflammatory cytokines (TNF-α, IL-1β and IL-6), chemokines (CXCL8-11, CCL20) and S100 proteins, which can in turn activate innate immune cells, leading to a vicious cycle of inflammation thereby maintaining and aggravating psoriasis. As such, IL-17A plays an important bridging role in the inflammatory circuit of innate and adaptive immune against psoriasis. Certainly, the IL-23/IL-17A inflammatory axis is only one important part of the pathologic processes of psoriasis. Recent studies have found that γδ T cells and macrophages could also secrete IL-17A and promote the pathogenesis of psoriasis (Cai, Y, Fleming, C. & Yan, J. Dermal gammadelta T cells—a new player in the pathogenesis of psoriasis. International immunopharmacology 16, 388-391, doi:10.1016/j.intimp.2013.02.018 (2013)), and Th22 cells could secrete IL-22 and aggravate the development of psoriasis, with the remaining pathological links yet to be found (Benham, H. et al. Th17 and Th22 cells in psoriatic arthritis and psoriasis. Arthritis research & therapy 15, R136, doi:10.1186/ar4317 (2013)).
1.2 Current Treatment Status for Autoimmune Psoriasis
At present, the treatment of psoriasis is classified mainly in accordance with the severity of the disease to be treated. Mild to moderate psoriasis is often treated with topical administration of drugs such as glucocorticoids, vitamin D3 analogues, vitamin A acid, anthralin, tar-based drugs, etc. For conditions in which the topical drugs are ineffective or for severe psoriasis, especially erythrodermic psoriasis, generalized pustular psoriasis and arthropathic psoriasis etc., a systemic dosing therapy is often used, such as intramuscular injection or intravenous infusion of small molecule immunosuppressive agents such as methotrexate and cyclosporine. In recent years, some biological formulations have also been approved by FDA for the treatment of severe psoriasis, such as monoclonal antibodies (mAbs) targeting T cell CD2 or CD11a, TNF-α, IL-12 and IL-23 (Table 1).
TABLE 1Recent progress in research and development of psoriasis drugsNamePharma CompanyTypeTargetR & D ProgressAlefaceptBiogenmAbT cell (CD2)approved in 2003EfalizumabGenentechmAbT cell (CD11a)approved in 2003, andrecalled in 2009EtanerceptAmgenmAbTNF-αapproved in 2004InfliximabJanssen BiotechmAbTNF-αapproved in 2006AdalimumabAbbottmAbTNF-αapproved in 2008UstekinumabJohnson, JanssenmAbp40approved in 2009Biotechnology(IL-12&IL-23)BriakinumabAbbottmAbp40Clinical phase 3(IL-12&IL-23)completedTildrakizumabMerckmAbIL-23p19Clinical phase 3GuselkumabJanssen BiotechmAbIL-23p19Clinical phase 3AMG-139AmgenmAbIL-23p19Clinical phase 2BI-655066Boehringer IngelheimmAbIL-23p19Clinical phase 2LY-3074828Eli LillymAbIL-23p19Clinical phase 1ABT-122Abbott and Abb ViemAbIL-17A & TNFapproved in 2009SecukinumabNovartismAbIL-17Aapproved in 2015BrodalumabAmgenmAbIL-17RAClinical phase 3IxekizumabEli LillymAbIL-17AClinical phase 3ApremilastCelgenesmallPDE4approved in 2014moleculeAN2728AnacorsmallPDE4Clinical phase 2moleculeTofacitinibPfizersmallJAKClinical phase 3moleculeIM0-3100IderasmallTLR7 & 9Clinical phase 2molecule
These biological formulations have significant efficacy but unknown safety, which need long-term clinical investigation. In addition, major pharmaceutical companies are also actively developing mAb drugs targeting IL-23, IL-17 or IL-17 receptors, among which is Secukinumab, the first IL-17 mAb in the world, developed by Novotis and approved by EU in early 2015. Unfortunately, for safety reasons, patients of mild to moderate degree cannot use these mAb drugs in a short term. In addition to the macromolecular monoclonal antibody drugs, some small molecule inhibitors have also been approved by FDA for the treatment of psoriasis, such as oral small molecule inhibitors targeting PDE4, and also small molecule inhibitors targeting TLR7 & 9 and JAK currently under clinical trials.
2 NF-κB family and its member c-Rel
2.1 NF-κB family and its members
The relationship between NF-κB family and its member c-Rel and autoimmune diseases has been reported in current fundamental researches. The NF-κB family of mammals is consisted of five members: c-Rel, RelA (p65), RelB, NF-κB1 (p50/p105), and NF-κB2 (p52/p100). The amino terminus of these proteins has a highly-conserved domain consisted of about 300 amino acid residues, referred to as the Rel homologous domain (RHD). This homologous domain functions in dimerization, interaction with IκB, nuclear localization, and binding to DNA. In contrast, the carboxyl terminus of these proteins is not conserved, and c-Rel, RelA and RelB have a transactivation domain at their carboxy terminus.
Newly synthesized NF-κBs are usually in homologous or heterologous dimeric forms that are bound to IκB and stored in the cytoplasm in an inactive state. Till now, nine members of the mammalian IκB family have been found: IκBα, IκBβ, IκBε, IκBζ, IκBη, BCL-3, IkB-NS, p100 (p52 precursor protein), and p105 (p50 precursor protein). These proteins prevent NF-κB from migrating into the nucleus mainly by covering the nuclear localization sequences of NF-κB family members. Some receptors, including receptors of tumor necrosis factor, IL-1 and nerve growth factor (NGF), T-cell and B-cell antigen receptors, and Toll-like receptors, can activate NF-κB upon binding to corresponding ligands. The activation of NF-κB requires IκB phosphorylation mediated by IκB kinases (IKK) and subsequent IκB protein degradation or processing (e.g., p100). Once NF-κB is activated, it enters the nucleus in a free dimeric form and binds to 9-10 base pairs in the promoter of the target gene to regulate the expression of the gene. The activated NF-κB can be down-regulated through a variety of mechanisms (such as feedback pathway), bound with newly synthesized IκB, and stored in the cytoplasm in a resting state.
2.2 C-Rel is Closely Related to the Development of Autoimmune Diseases
Studies have shown that NF-κB plays a critical role in the development of autoimmune diseases in addition to its role in maintaining physiological functions and pathological states. In mice and humans, the onset of type 1 diabetes is often accompanied by a high level activation of NF-κB in dendritic cells and mononuclear cells, as well as infiltration of these cells in tissues. Inhibiting activation of NF-κB can effectively inhibit the occurring of type 1 diabetes in NOD mice, CD1 mice and C57BL/6 mice (Campbell, I. K., Gerondakis, S., O'Donnell, K. & Wicks, I. P. Distinct roles for the NF-kappaB1 (p50) and c-Rel transcription factors in inflammatory arthritis. The Journal of clinical investigation 105, 1799-1806, doi:10.1172/jci8298 (2000)). To directly investigate the role of NF-κB in the development of autoimmune diseases, Liou et al. (together with the laboratory of the present inventors) used c-Rel and p50 knocked-out mice as experimental subjects to investigate the incidence of type I diabetes mellitus, arthritis and encephalomyelitis (B. A. Hilliard, N. Mason, L. Xu, J. Sun, S. E. Lamhamedi-Cherradi, H. C. Liou, C. Hunter, Y. H. Chen, Critical roles of c-Rel in autoimmune inflammation and helper T cell differentiation. The Journal of clinical investigation 110, 843-850 (2002); published online EpubSep (10.1172/jci15254); S. E. Lamhamedi-Cherradi, S. Zheng, B. A. Hilliard, L. Xu, J. Sun, S. Alsheadat, H. C. Liou, Y. H. Chen, Transcriptional regulation of type I diabetes by NF-kappa B. Journal of immunology (Baltimore, Md.: 1950) 171, 4886-4892 (2003); Q. Ruan, V. Kameswaran, Y. Zhang, S. Zheng, J. Sun, J. Wang, J. DeVirgiliis, H. C. Liou, A. A. Beg, Y. H. Chen, The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis. The Journal of experimental medicine 208, 2321-2333 (2011); published online EpubOct 24 (10.1084/jem.20110462)). These studies discovered that c-Rel knocked-out mice developed normally with an immune system of a normal constitution, not suffering from spontaneous infectious diseases, and when challenged with high-dose pathogens, they could clear pathogens with a normal or slight reduced clearing capacity. Meanwhile, C-Rel knocked-out mice were resistant to autoimmune diseases such as encephalomyelitis, type 1 diabetes, and arthritis. These phenomena suggest that c-Rel is closely related to the development of autoimmune diseases.
c-Rel and Inflammatory Response in Autoimmune Diseases
APC, T cells, and B cells in mice having autoimmune diseases are often associated with excessive activation of c-Rel, and the resistance of c-Rel knocked-out mice to the incidence of autoimmune diseases suggests that c-Rel plays a critical regulatory role in the pathogenesis of autoimmune diseases. Researches have shown that c-Rel was involved in regulating the expression of a number of inflammatory factors in antigen-presenting cells as well as the differentiation and development of Th17, mainly in the following aspects.
3.1 c-Rel Directly Regulates the Expression of a Number of Proinflammatory Factors in Antigen Presenting Cells
Both dendritic cells and macrophages are specialized antigen presenting cells in the innate immune system of the body, which are capable of initiating the adaptive immune system. The inventors of the present invention found in a study on bone marrow-derived dendritic cells (BMDC) that the expression of IL-23p19 in c-Rel knocked-out BMDC was significantly lower than that in wild-type BMDC (Carmody, R. J., Ruan, Q., Liou, H. C. & Chen, Y. H. Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells. Journal of immunology (Baltimore, Md.: 1950) 178, 186-191 (2007)). It was found by in vitro luciferase assay (luciferase), gel migration assay (EMSA) and in vivo chromatin immunoprecipitation (ChIP) techniques that when Toll-like receptors (TLRs) conducted signal transduction, c-Rel could specifically bind to the IL-23p19 gene promoter at two binding sites and form a enhancer together with additional transcription factors to directly regulate IL-23p19 expression. Most importantly, when only the additional transcription factors (the p19 promoter also has potential binding sites for AP-1, C/EBP, and IRF) bound to the p19 gene promoter, the p19 gene was not activated, indicating that the expression of the IL-23p19 gene was completely dependent on c-Rel. Furthermore, Nicola Mason et al. found that during inflammatory stimulation (e.g., LPS), c-Rel in macrophages and dendritic cells directly regulated, in the form of a c-Rel/p50 heterodimer, the expression of IL-12p40 (N. Mason, J. Aliberti, J. C. Caamano, H. C. Liou, C. A. Hunter, Cutting edge: identification of c-Rel-dependent and -independent pathways of IL-12 production during infectious and inflammatory stimuli. Journal of immunology (Baltimore, Md.: 1950) 168, 2590-2594 (2002)). In addition, researches showed that c-Rel was also involved in regulating the expression of IL-6 and other pro-inflammatory factors in APC (J. R. Tumang, C. Y. Hsia, W. Tian, J. F. Bromberg, H. C. Liou, IL-6 rescues the hyporesponsiveness of c-Rel deficient B cells independent of Bcl-xL, Mcl-1, and Bcl-2. Cellular immunology 217, 47-57 (2002)).
3.2 c-Rel Directly or Indirectly Regulate the Differentiation and Development of Th17 Cells
Previously, it was commonly recognized by researchers that Th1 cells were the primary pathogenic cells in the pathogenesis of autoimmune diseases. However, in recent years, it has been discovered in more in-depth studies that Th17 cells played a more critical pathogenic role in a variety of autoimmune diseases, including multiple sclerosis, psoriasis, rheumatoid arthritis and the like. Unlike Th1 cells, Th17 cells mainly produce cytokines such as IL-17A, IL-17F and IL-22, and play a role in inflammatory diseases and combating bacterial infections. Th17 lineage specific factors include RORγt, RORα and STAT3.
c-Rel affects the development and differentiation of Th17 in two aspects. In one aspect, as discovered by the present inventors, c-Rel indirectly regulates Th17 cell development by directly regulating IL-23 production in antigen presenting cells (Carmody, R. J., Ruan, Q., Liou, H. C. & Chen, Y. H. Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells. Journal of immunology (Baltimore, Md.: 1950) 178, 186-191 (2007)). In aother aspect, as found by the inventors, c-Rel can also directly regulate Th17 differentiation in T cells (Ruan, Q. et al. The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis. The Journal of experimental medicine 208, 2321-2333, doi:10.1084/jem.20110462 (2011)). When the inventors of the present application, Ruan, Q et al., used an anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody to stimulate the CD4+ T cells isolated from c-Rel knocked-out mice, the expression of IL-17A messenger RNAs (mRNAs) and IL-17A proteins was significantly reduced in comparison with that in wild-type cells; and when the CD4+ T cells isolated from c-Rel knocked-out mice were cultured and stimulated in vitro, the number of Th17 was found to be reduced up to 70% (Ruan, Q. et al. The Th17 immune response is controlled by the Rel-RORgamma-RORgamma T transcriptional axis. The Journal of experimental medicine 208, 2321-2333, doi:10.1084/jem.20110462 (2011); Ruan, Q. et al. Development of Foxp3(+) regulatory t cells is driven by the c-Rel enhanceosome. Immunity 31, 932-940, doi:10.1016/j.immuni.2009.10.006 (2009)). Further studies showed that the expression of RORγ and RORγT was significantly decreased in c-Rel knocked-out T cells, while the reestablishment of RORγ and RORγT could restore the differentiation defects of Th17 in c-Rel knocked-out T cells. In addition, with the chromatin immunoprecipitation technique, Ruan, Q et al. found that c-Rel/p65 regulated the expression of RORγT and RORγ mRNAs by respectively binding and activating two different Rorγ promoters. Based on the results of the above studies, the inventors of the present application, Ruan, Q et al., proposed a Th17 differentiation theory: on the CD4+ T cells, when TCR, costimulatory molecules, and cytokine receptors such as IL-1, IL-23, and IL-6 binding with the corresponding ligands (Th17 differentiation conditions), activation signals are transducted into the cell, and multiple transcription factors were released into the nucleus. In this process, the free c-Rel/p65 dimer binds to different promoters of the Rorγ gene and interacts with other transcription factors (such as NFAT and Stat) that are released into the nucleus to form Rorγ-specific enhancers, initiating the transcription of the Rorγ gene, which drives the Th17 cells to differentiate. It is noteworthy that the c-Rel/p65 transcription factors are the only transcription factors currently found to bind to the Rorγ gene promoters and activate transcription, and other transcription factors binding to the Rorγ gene promoters remain to be discovered.
4 Treatment of Autoimmune Psoriasis by Targeting the Entire NF-κB Family
Theoretically, drugs targeting the entire NF-κB family may be effective in treating autoimmune psoriasis, such as protease inhibitors (e.g., the FDA-approved PS-341), NF-κB decoy oligonucleotides, NBD polypeptides, glucocorticoids and the like (Vanderlugt, C. L., Rahbe, S. M., Elliott, P. J., Dal Canto, M. C. & Miller, S. D. Treatment of established relapsing experimental autoimmune encephalomyelitis with the proteasome inhibitor PS-519. Journal of autoimmunity 14, 205-211, doi:10.1006/jaut.2000.0370 (2000); May, M. J. et al. Selective inhibition of NF-kappaB activation by a peptide that blocks the interaction of NEMO with the IkappaB kinase complex. Science 289, 1550-1554 (2000); De Stefano, D. Oligonucleotides decoy to NF-kappaB: becoming a reality? Discovery medicine 12, 97-105 (2011)). However, most of the NF-κB family proteins are commonly expressed in many cells in the body and are associated with the maintenance of normal physiological functions such as congenital and adaptive immunity regulation during infection, inflammatory response, anti-apoptosis, cell proliferation and the like. As a result, these drugs have severe side effects, poor specificity, and can only be used in a short term to control acute allergic reactions. Thus, those drugs that target the entire NF-κB family can not be used to treat chronic inflammatory diseases, such as autoimmune psoriasis.